The biogenetic sulfidation of zero-valent iron (BS-ZVI) by sulfate-reducing bacteria (SRB) has been demonstrated to enhance the reactivity of ZVI. However, long-term performance of BS-ZVI and related mechanism were still unknown. Therefore, columns containing sponge iron and SRB are built to prepare BS-ZVI in-situ and study its long-term performance. Over 80 % of NO3‾ was reduced to NH4+ by in-situ BS-ZVI within 140 days, which was higher than the sole ZVI treatment (40 %-60 %). The bonding of ZVI and FeSx was in-situ firstly and finally loaded on ZVI. The reduction of Fe(III) by S(−II) and SRB contributed to the formation of FeSx, which improved the electrons transfer. Moreover, BS-ZVI enhanced the enzymes activity of SRB, thus accelerating the metabolic transformation of lactic acid to acetic acid. The accumulation of acetic acid enhanced the removal efficiency of NO3‾ through the dissolution of passivation layer. Overall, this study demonstrated a reactivity enhancement of ZVI through biogenetic sulfidation, which provided a new alternative method for the remediation of groundwater.